Folate

Background

Serum folate levels have garnered tremendous interest because of its role in preventing certain type of birth defects and more recently, as a prophylactic role in preventing spontaneous abortions.

OUTLINE

Reference Methods

Clinical Utility

Interfering Diseases or Substances that Alter Levels

Commonly Used Terms

Internet Links

REFERENCE METHODS CHARACTERIZATION

Comparison of Five Automated Serum and Whole Blood Folate Assays

William E. Owen, MT(ASCP),1 and William L. Roberts, MD, PhD
Am J Clin Pathol 2003;120:121-126 Abstract quote
Serum and whole blood folate measurements are used to establish folate deficiency. Most methods used in clinical laboratories are automated, nonisotopic methods that use folate-binding protein.

Linearity, imprecision, and method comparison studies, including serum and whole blood hemolysates, were performed with the Access, Advia Centaur, ARCHITECT i2000, Elecsys 2010, and IMMULITE 2000 methods. The QuantaPhase II radioassay served as the comparison method. (Proprietary information is given in the text.)

The Access and IMMULITE 2000 methods had higher systematic errors in linearity studies than the other 3 methods. The imprecision of all methods was acceptable (coefficient of variation, <10%) even at low folate concentrations with the exception of the Elecsys 2010 (coefficient of variation, 16%). Method comparison studies using serum samples revealed calibration differences between the Access and Elecsys 2010 methods and the comparison method. Method comparison studies using whole blood samples showed poorer agreement between each of the automated methods and the comparison method than was seen with serum samples.

The ARCHITECT i2000 folate assay demonstrated the best analytic performance. The poor agreement seen with whole blood hemolysates likely is due to calibration differences and differences in hemolysate preparation conditions.

Folate assays: serum or red cell?

Phekoo K, Williams Y, Schey SA, Andrews VE, Dudley JM, Hoffbrand AV.

Guy's and St Thomas' Trust, London.

J R Coll Physicians Lond 1997 May-Jun;31(3):291-5 Abstract quote
Tests for folate and vitamin B12 deficiency are frequently requested by clinicians in many different specialties. An audit of folate assay methodology was undertaken to establish the number of tests and types of assay performed in different centres, and to analyse the indications for these investigations, with a view to advising on the most appropriate assay for use in the laboratory. A questionnaire was sent to 30 centres, 24 (80%) of which participated in the audit. The types of folate assay performed, number of requests, reference range and method of analysis differed between centres. The major specialty users of the service were general practitioners, general physicians and geriatricians.

A detailed analysis of 1,259 consecutive requests for folate assays from a single representative laboratory showed a significant correlation between serum and red cell folate levels (r = 0.49, p < 0.001). However, in patients with low serum folate, there was no correlation with red cell folate in the absence of macrocytosis. The major indication for folate analysis was for haematological abnormalities but 36% of cases were for nonspecific indications. A haematologist with an interest in folate metabolism was invited to moderate the results at an audit meeting of haematologists.

The consensus was that the most appropriate screening test for folate deficiency is the serum assay, which can be combined easily with vitamin B12 assay.

Serum folate levels among women attending family planning clinics--Georgia, 2000.

Than LC, Watkins M, Daniel KL.

MMWR Recomm Rep 2002 Sep 13;51(RR-13):4-8 Abstract quote
Since 1998, serum folate levels have increased nationally after mandatory fortification of cereal grain products with folic acid. Whether serum folate levels have increased among all women has not been well-studied. Identifying characteristics of women with lower serum folate levels would also be helpful in designing educational campaigns.

Data for this report were collected during January 2000-January 2001. During 2000, blood samples were collected from 1,059 women aged 18-45 years who attended six family planning clinics in Georgia and analyzed for serum folate levels. This sample included women aged 18-25 years (60%), black women (41%), and women who had a high school education or less (49%). The median serum folate level (8.9 ng/mL) among this population was lower than the median of women of childbearing age (13.0 ng/mL) who participated in the 1999-2000 National Health and Nutrition Examination Survey (NHANES). In logistic regression analysis, women who were black (odds ratio [OR] = 2.4; 95% confidence interval [CI] = 1.48, 3.96), who smoked (OR = 2.1; 95% CI = 1.26, 3.43), or who used Depo-Provera contraceptive injection (manufactured by Pharmacia Corporation, Peapack, New Jersey) (OR = 2.3; 95% CI = 1.15, 4.62) were more likely to be ranked in the lowest quartile (< or = 62 ng/mL) of serum folate concentrations when compared with the highest quartile (> 12.4 ng/mL).

Women who consumed cereal regularly (OR = 0.4; 95% CI = 0.26, 0.62) or folic acid supplements (OR = 0.2; 95% CI = 0.09, 0.30) were the least likely to be in the lowest serum folate quartile.

This study indicates that certain women are at greater risk for having lower serum folate levels, including women who are black, smokers, Depo-Provera users, and those less likely to eat cereal regularly or to take folic acid supplements. In Georgia, these data are useful in defining target populations (e.g., black women and smokers) for folic acid education campaigns because public health officials can develop contextually appropriate messages and outreach approaches for targeting women for folic acid interventions. Ongoing surveillance of serum folate status among women can guide future intervention efforts.

CLINICAL UTILITY CHARACTERIZATION

HOMOCYSTEINE

An Ontario-wide study of vitamin B12, serum folate, and red cell folate levels in relation to plasma homocysteine: is a preventable public health issue on the rise?.

Ray JG, Cole DE, Boss SC.

Department of Medicine, Women's College Hospital, University of Toronto, Toronto, Canada.
Clin Biochem 2000 Jul;33(5):337-43 Abstract quote
BACKGROUND: Plasma homocysteine has been reported to be useful in the evaluation of patients with suspected vitamin B12 or folate deficiency. In November 1998, Canada began its mandatory fortification of all flour, and some corn and rice products, with folic acid. We evaluated the status of folate and vitamin B12 in Ontario since this fortification program began, and also studied the role of plasma homocysteine in the assessment of vitamin B12 deficiency since that time.

METHODS: A retrospective cross-sectional study design was performed using a community database of all Ontario samples analyzed by MDS Laboratories, a major provider of diagnostic laboratory services in Canada. All consecutive single-patient fasting samples for plasma homocysteine collected between January 1 and September 30, 1999 were included, as well as corresponding red cell folate and serum B12 concentrations. Data for serum folate were included when available. Descriptive statistics included the arithmetic and geometric means for each measure, as well as the lower and upper centile values. After excluding cases with a concomitant serum creatinine > 120 micromol/L or red cell folate < 215 nmol/L, we established the test properties of a plasma homocyteine level of 15 micromol/L or greater for the diagnosis of "low" (< 120 pmol/L) or "indeterminate" (i.e., between 120 and 150 pmol/L) serum vitamin B12 concentrations.

RESULTS: The mean age of all subjects was 58.4 years (95% CI 57.4 to 59.4). Plasma homocysteine samples were obtained from 403 males (56.7%) and 308 females. The geometric mean homocysteine concentration for the entire population was 8.3 micromol/L, and was significantly higher among males (9.3 micromol/L) than females (8.3 micromol/L) (unpaired t-test: 2-p < 0.0001). The geometric mean serum folate concentration was significantly higher in females (35.8 nmol/L) than males (33.6 nmol/L) (2-p < 0.0001), as were the mean red cell folate levels (females 966.8 nmol/L, males 949.3 nmol/L; 2-p < 0.0001). Serum vitamin B12 concentrations were available for 692 subjects, with a geometric mean of 322.0 pmol/L. Again, mean vitamin B12 was higher in females (332.5 pmol/L) than males (314.3 pmol/L) (2-p < 0.0001). The fifth centile for vitamin B12 was 134.6 pmol/L. A plasma homocysteine concentration > 15 micromol/L did not discriminate between cobalamin concentrations below versus above 120 pmol/L (positive and negative predictive values 7.4% and 97.2%, respectively), nor did it discriminate "indeterminate" B12 levels between 120 and 150 pmol/L (positive and negative predictive values 6.3% and 94.0%, respectively).

CONCLUSION: In a large select group of Ontarians, serum and red cell folate concentrations appear to be higher than expected, possibly due to a recent national folate fortification programme; cobalamin levels are no higher than expected. Given our inability to detect mild B12 deficiency using such indicators as plasma homocysteine, and considering the substantial growth in the elderly segment of the Canadian population, occult cobalamin deficiency could become a common disorder. Accordingly, we recommend either consideration of the addition of vitamin B12 to the current folate fortification programme, and/or the development of better methods for the detection of cobalamin deficiency.

SPONTANEOUS ABORTION

Preconception folate and vitamin B(6) status and clinical spontaneous abortion in Chinese women.

Ronnenberg AG, Goldman MB, Chen D, Aitken IW, Willett WC, Selhub J, Xu X.

Department of Environmental Health, Program for Population Genetics, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Obstet Gynecol 2002 Jul;100(1):107-13 Abstract quote
OBJECTIVE: To assess the association between preconception homocysteine and B vitamin status and risk of clinical spontaneous abortion in women from Anqing, China.

METHODS: All women were aged 21-34 years, had never smoked, and were primigravid. Patients (n = 49) were women with a clinically recognized pregnancy who experienced a fetal death before 100 days' gestation. Controls (n = 409) were women who maintained a pregnancy that ended in a live birth. Homocysteine, folate, and vitamins B(6) and B(12) concentrations were measured in plasma obtained before conception.

RESULTS: Mean vitamin B(6) concentration was lower in patients than in controls (34.0 versus 37.9 nmol/L, P =.04). In addition, the risk of spontaneous abortion tended to increase with decreasing plasma vitamin B(6) and folate concentration (P for trend =.06 and.07, respectively), although the significance of these trends was further reduced in logistic models that included age, body mass index, and both vitamins. The risk of spontaneous abortion was four-fold higher among women with suboptimal plasma concentrations of both folate and vitamin B(6) (folate less than or equal to 8.4 nmol/L and vitamin B(6) less than or equal to 49 nmol/L) than in those with higher plasma concentrations of both vitamins (odds ratio 4.1, 95% confidence interval 1.2, 14.4). Homocysteine and vitamin B(12) status were not associated with spontaneous abortion risk.

CONCLUSION: Suboptimal preconception folate and vitamin B(6) status, especially when they occur together, may increase the risk of clinical spontaneous abortion. Additional prospective studies are needed to confirm these findings and to determine whether antenatal B vitamin supplementation reduces spontaneous abortion risk.

Plasma folate levels and risk of spontaneous abortion.

George L, Mills JL, Johansson AL, Nordmark A, Olander B, Granath F, Cnattingius S.

Department of Medical Epidemiology, Karolinska Institutet, PO Box 281, SE-171 77 Stockholm, Sweden.
JAMA 2002 Oct 16;288(15):1867-73 Abstract quote
CONTEXT: Both folate deficiency and folic acid supplements have been reported to increase the risk of spontaneous abortion. The results are inconclusive, however, and measurements of folate have not been available in all studies.

OBJECTIVE: To study the association between plasma folate levels and the risk of spontaneous abortion.

DESIGN, SETTING, AND POPULATION: Population-based, matched, case-control study of case women with spontaneous abortion and control women from January 1996 through December 1998 in Uppsala County, Sweden. Plasma folate measurements were available for 468 cases and 921 controls at 6 to 12 gestational weeks.

MAIN OUTCOME MEASURE: Risk of spontaneous abortion vs maternal plasma folate level.

RESULTS: Compared with women with plasma folate levels between 2.20 and 3.95 ng/mL (5.0 and 8.9 nmol/L), women with low (</=2.19 ng/mL [</=4.9 nmol/L]) folate levels were at increased risk of spontaneous abortion (adjusted odds ratio [OR], 1.47; 95% confidence interval [CI], 1.01-2.14), whereas women with higher folate levels (3.96-6.16 ng/mL [9.0-13.9 nmol/L] and >/=6.17 ng/mL [>/=14.0 nmol/L]) showed no increased risk of spontaneous abortion (OR, 0.84; 95% CI, 0.59-1.20; and OR, 0.74; 95% CI, 0.47-1.16, respectively). Low folate levels were associated with a significantly increased risk when the fetal karyotype was abnormal (OR, 1.95; 95% CI, 1.09-3.48) but not when the fetal karyotype was normal (OR, 1.11; 95% CI, 0.55-2.24) or unknown (OR, 1.45; 95% CI, 0.90-2.33).

CONCLUSION: Low plasma folate levels were associated with an increased risk of early spontaneous abortion.

INTERFERING DISEASES OR SUBSTANCES CHARACTERIZATION

ALTERED LEVELS

Drugs and folate metabolism.

Lambie DG, Johnson RH.

Drugs 1985 Aug;30(2):145-55 Abstract quote
Folates are a group of compounds which are required in the diet and are important in DNA, amino acids and possibly also amine metabolism. The biologically active folates are in the tetrahydro form. Tetrahydrofolates are produced from unreduced dietary folates by the enzyme dihydrofolate reductase.

A number of drugs such as aminopterin, methotrexate (amethopterin), pyrimethamine, trimethoprim and triamterene act as folate antagonists and produce folate deficiency by inhibiting this enzyme. With other drugs which produce low serum and tissue concentrations of folate such as anticonvulsants, antituberculosis drugs, alcohol and oral contraceptives, the mechanism of this effect is uncertain. Possible mechanism include reduced absorption, prevention of release of folate from tissue stores, altered plasma protein binding, or increased folate metabolism in the liver.

Treatment with folic acid antagonists such as methotrexate readily causes megaloblastic anaemia; this can be prevented by therapy with folinic acid (5-formyltetrahydrofolate). The role of other drugs in producing megaloblastic anaemia is less certain, e.g. it occurs in less than 0.75% of patients receiving anticonvulsants. The possible neurological and psychiatric effects of folate deficiency are also uncertain.

However, in patients with folate deficiency who have neuropsychiatric symptoms, neuropathy or myelopathy, and normal vitamin B12 levels, it may be of value to try therapy with folic or folinic acid.

INCREASE LEVELS

DIALYSIS

Homocysteine, vitamin B12, and serum and erythrocyte folate in peritoneal dialysis and hemodialysis patients.

De Vecchi AF, Bamonti-Catena F, Finazzi S, Campolo J, Taioli E, Novembrino C, Colucci P, Accinni R, De Franceschi M, Fasano MA, Maiolo AT.

Divisione di Nefrologia e Dialisi, IRCCS Ospedale Maggiore, Milano, Italy. suggests that levels of folate within the reference interval are inadequate for dialysis patients.
Perit Dial Int 2000 Mar-Apr;20(2):169-73 Abstract quote
BACKGROUND: Plasma homocysteine (Hcy) is an independent risk factor for cardiovascular disease. High levels of plasma Hcy have been observed in end-stage renal disease patients. Few studies have compared peritoneal dialysis (PD) and hemodialysis (HD) patients and few data are available on erythrocyte folate (ery-F) levels in dialysis patients.

OBJECTIVES: To evaluate plasma Hcy concentrations, vitamin B12 (B12), and folate status in dialysis patients; to analyze the possible causes of high Hcy levels; to follow up changes in folate and B12 concentrations after 6 months. DESIGN: A cross-sectional observational study. SETTING: Nephrology division and laboratory of hematology in a university and clinical research hospital. PATIENTS: The study included 82 patients treated with PD for 37 + 37 months and 70 patients treated with HD for 136 + 95 months. LABORATORY METHODS: Plasma Hcy was measured by the immunoenzymatic IMx Hcy FPIA method (Abbott Laboratories, Diagnostic Division, Abbott Park, IL, U.S.A.), serum folate (s-F) and ery-F by the Stratus folate fluorometric enzyme-linked assay, and B12 by the Stratus vitamin B12 fluorometric enzyme-linked assay (DADE-Behring, Newark, DE, U.S.A.). RESULTS: Ninety-six percent of PD and 97% of HD patients had Hcy levels above the cutoff (13.5 micromol/L). Homocysteine level was higher in HD than in PD patients, while the prevalence of hyperhomocysteinemia was similar with the two techniques. Erythrocyte folate was significantly higher in PD (1333 +/- 519 pmol/L) than in HD (1049 +/-511 pmol/L, p < 0.01). Statistically significant correlations were observed between Hcy and B12, s-F, ery-F, and dialysis duration. Multivariate analysis showed a strong correlation between s-F and Hcy. After 6 months there were no differences in Hcy, B12, s-F, and ery-F levels. CONCLUSIONS: Plasma Hcy levels were high in more than 95% of our dialysis patients, with no relation to the type of dialysis. Vitamin B12 and folate were normal in the majority of our patients. However, serum folate was the major determinant of Hcy levels. Such a relation between Hcy and folate suggests that levels of folate within the reference interval are inadequate for dialysis patients.

DECREASE LEVELS

HOMOCYSTEINE

Elevated serum homocysteine as a predictor for vitamin B12 or folate deficiency.

Curtis D, Sparrow R, Brennan L, Van der Weyden MB.

Haematology Unit, Alfred Group of Hospitals Pathology Service, Alfred Hospital, Prahran, Victoria, Australia.
Eur J Haematol 1994 Apr;52(4):227-32 Abstract quote
Tissue deficiency of vitamin B12 and folate results in an increase in serum homocysteine (sHcy). We have measured sHcy in patients with reduced serum vitamin B12 and/or red cell folate (RCF) to determine its usefulness as a discriminant for the diagnostic interpretation of reduced vitamin levels. Of 3846 patients who had serum vitamin B12 and RCF assayed, 335 (9%) had reduced vitamin levels.

Multivariate analysis showed a significant association between sHcy and serum creatinine (p = 0.0001), positive intrinsic factor (IF) antibody or neutrophil hypersegmentation (NHS) (p = 0.001), increased MCV (p = 0.014) and low RCF (p = 0.025) but no relationship with the level of serum vitamin B12 or haemoglobin. After censoring the patients with renal impairment (n = 54), the distribution of the remaining 72 patients with elevated sHcy was 37/151 (25%) with low serum vitamin B12 with or without low RCF and 35/130 (27%) with low RCF alone. sHcy correctly identified response to vitamin therapy in 33/35 (94%) patients who had adequate parameters to assess response. The positive predictive values of IF antibody/NHS, macrocytosis and/or low RCF for elevated sHcy were 100% and 34% respectively. Twenty-four percent of patients with a low serum vitamin B12 and elevated sHcy had no abnormal haematologic parameters as determined by the routine laboratory staff.

These data suggest that the usefulness of measuring sHcy in a routine diagnostic setting is limited and a careful review of the peripheral blood for macrocytosis and NHS plus determination of RCF may be a more cost-effective process than sHcy assay in most instances to determine the presence of tissue deficiency.

Henry JB. Clinical Diagnosis and Management by Laboratory Methods. Twentieth Edition. WB Saunders. 2001.

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Homocysteine

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Last Updated 7/9/2003

REFERENCE METHODS	CHARACTERIZATION
Comparison of Five Automated Serum and Whole Blood Folate Assays William E. Owen, MT(ASCP),1 and William L. Roberts, MD, PhD	Am J Clin Pathol 2003;120:121-126 Abstract quote Serum and whole blood folate measurements are used to establish folate deficiency. Most methods used in clinical laboratories are automated, nonisotopic methods that use folate-binding protein. Linearity, imprecision, and method comparison studies, including serum and whole blood hemolysates, were performed with the Access, Advia Centaur, ARCHITECT i2000, Elecsys 2010, and IMMULITE 2000 methods. The QuantaPhase II radioassay served as the comparison method. (Proprietary information is given in the text.) The Access and IMMULITE 2000 methods had higher systematic errors in linearity studies than the other 3 methods. The imprecision of all methods was acceptable (coefficient of variation, <10%) even at low folate concentrations with the exception of the Elecsys 2010 (coefficient of variation, 16%). Method comparison studies using serum samples revealed calibration differences between the Access and Elecsys 2010 methods and the comparison method. Method comparison studies using whole blood samples showed poorer agreement between each of the automated methods and the comparison method than was seen with serum samples. The ARCHITECT i2000 folate assay demonstrated the best analytic performance. The poor agreement seen with whole blood hemolysates likely is due to calibration differences and differences in hemolysate preparation conditions.
Folate assays: serum or red cell? Phekoo K, Williams Y, Schey SA, Andrews VE, Dudley JM, Hoffbrand AV. Guy's and St Thomas' Trust, London.	J R Coll Physicians Lond 1997 May-Jun;31(3):291-5 Abstract quote Tests for folate and vitamin B12 deficiency are frequently requested by clinicians in many different specialties. An audit of folate assay methodology was undertaken to establish the number of tests and types of assay performed in different centres, and to analyse the indications for these investigations, with a view to advising on the most appropriate assay for use in the laboratory. A questionnaire was sent to 30 centres, 24 (80%) of which participated in the audit. The types of folate assay performed, number of requests, reference range and method of analysis differed between centres. The major specialty users of the service were general practitioners, general physicians and geriatricians. A detailed analysis of 1,259 consecutive requests for folate assays from a single representative laboratory showed a significant correlation between serum and red cell folate levels (r = 0.49, p < 0.001). However, in patients with low serum folate, there was no correlation with red cell folate in the absence of macrocytosis. The major indication for folate analysis was for haematological abnormalities but 36% of cases were for nonspecific indications. A haematologist with an interest in folate metabolism was invited to moderate the results at an audit meeting of haematologists. The consensus was that the most appropriate screening test for folate deficiency is the serum assay, which can be combined easily with vitamin B12 assay.
Serum folate levels among women attending family planning clinics--Georgia, 2000. Than LC, Watkins M, Daniel KL.	MMWR Recomm Rep 2002 Sep 13;51(RR-13):4-8 Abstract quote Since 1998, serum folate levels have increased nationally after mandatory fortification of cereal grain products with folic acid. Whether serum folate levels have increased among all women has not been well-studied. Identifying characteristics of women with lower serum folate levels would also be helpful in designing educational campaigns. Data for this report were collected during January 2000-January 2001. During 2000, blood samples were collected from 1,059 women aged 18-45 years who attended six family planning clinics in Georgia and analyzed for serum folate levels. This sample included women aged 18-25 years (60%), black women (41%), and women who had a high school education or less (49%). The median serum folate level (8.9 ng/mL) among this population was lower than the median of women of childbearing age (13.0 ng/mL) who participated in the 1999-2000 National Health and Nutrition Examination Survey (NHANES). In logistic regression analysis, women who were black (odds ratio [OR] = 2.4; 95% confidence interval [CI] = 1.48, 3.96), who smoked (OR = 2.1; 95% CI = 1.26, 3.43), or who used Depo-Provera contraceptive injection (manufactured by Pharmacia Corporation, Peapack, New Jersey) (OR = 2.3; 95% CI = 1.15, 4.62) were more likely to be ranked in the lowest quartile (< or = 62 ng/mL) of serum folate concentrations when compared with the highest quartile (> 12.4 ng/mL). Women who consumed cereal regularly (OR = 0.4; 95% CI = 0.26, 0.62) or folic acid supplements (OR = 0.2; 95% CI = 0.09, 0.30) were the least likely to be in the lowest serum folate quartile. This study indicates that certain women are at greater risk for having lower serum folate levels, including women who are black, smokers, Depo-Provera users, and those less likely to eat cereal regularly or to take folic acid supplements. In Georgia, these data are useful in defining target populations (e.g., black women and smokers) for folic acid education campaigns because public health officials can develop contextually appropriate messages and outreach approaches for targeting women for folic acid interventions. Ongoing surveillance of serum folate status among women can guide future intervention efforts.

CLINICAL UTILITY	CHARACTERIZATION
HOMOCYSTEINE
An Ontario-wide study of vitamin B12, serum folate, and red cell folate levels in relation to plasma homocysteine: is a preventable public health issue on the rise?. Ray JG, Cole DE, Boss SC. Department of Medicine, Women's College Hospital, University of Toronto, Toronto, Canada.	Clin Biochem 2000 Jul;33(5):337-43 Abstract quote BACKGROUND: Plasma homocysteine has been reported to be useful in the evaluation of patients with suspected vitamin B12 or folate deficiency. In November 1998, Canada began its mandatory fortification of all flour, and some corn and rice products, with folic acid. We evaluated the status of folate and vitamin B12 in Ontario since this fortification program began, and also studied the role of plasma homocysteine in the assessment of vitamin B12 deficiency since that time. METHODS: A retrospective cross-sectional study design was performed using a community database of all Ontario samples analyzed by MDS Laboratories, a major provider of diagnostic laboratory services in Canada. All consecutive single-patient fasting samples for plasma homocysteine collected between January 1 and September 30, 1999 were included, as well as corresponding red cell folate and serum B12 concentrations. Data for serum folate were included when available. Descriptive statistics included the arithmetic and geometric means for each measure, as well as the lower and upper centile values. After excluding cases with a concomitant serum creatinine > 120 micromol/L or red cell folate < 215 nmol/L, we established the test properties of a plasma homocyteine level of 15 micromol/L or greater for the diagnosis of "low" (< 120 pmol/L) or "indeterminate" (i.e., between 120 and 150 pmol/L) serum vitamin B12 concentrations. RESULTS: The mean age of all subjects was 58.4 years (95% CI 57.4 to 59.4). Plasma homocysteine samples were obtained from 403 males (56.7%) and 308 females. The geometric mean homocysteine concentration for the entire population was 8.3 micromol/L, and was significantly higher among males (9.3 micromol/L) than females (8.3 micromol/L) (unpaired t-test: 2-p < 0.0001). The geometric mean serum folate concentration was significantly higher in females (35.8 nmol/L) than males (33.6 nmol/L) (2-p < 0.0001), as were the mean red cell folate levels (females 966.8 nmol/L, males 949.3 nmol/L; 2-p < 0.0001). Serum vitamin B12 concentrations were available for 692 subjects, with a geometric mean of 322.0 pmol/L. Again, mean vitamin B12 was higher in females (332.5 pmol/L) than males (314.3 pmol/L) (2-p < 0.0001). The fifth centile for vitamin B12 was 134.6 pmol/L. A plasma homocysteine concentration > 15 micromol/L did not discriminate between cobalamin concentrations below versus above 120 pmol/L (positive and negative predictive values 7.4% and 97.2%, respectively), nor did it discriminate "indeterminate" B12 levels between 120 and 150 pmol/L (positive and negative predictive values 6.3% and 94.0%, respectively). CONCLUSION: In a large select group of Ontarians, serum and red cell folate concentrations appear to be higher than expected, possibly due to a recent national folate fortification programme; cobalamin levels are no higher than expected. Given our inability to detect mild B12 deficiency using such indicators as plasma homocysteine, and considering the substantial growth in the elderly segment of the Canadian population, occult cobalamin deficiency could become a common disorder. Accordingly, we recommend either consideration of the addition of vitamin B12 to the current folate fortification programme, and/or the development of better methods for the detection of cobalamin deficiency.
SPONTANEOUS ABORTION
Preconception folate and vitamin B(6) status and clinical spontaneous abortion in Chinese women. Ronnenberg AG, Goldman MB, Chen D, Aitken IW, Willett WC, Selhub J, Xu X. Department of Environmental Health, Program for Population Genetics, Harvard School of Public Health, Boston, Massachusetts 02115, USA.	Obstet Gynecol 2002 Jul;100(1):107-13 Abstract quote OBJECTIVE: To assess the association between preconception homocysteine and B vitamin status and risk of clinical spontaneous abortion in women from Anqing, China. METHODS: All women were aged 21-34 years, had never smoked, and were primigravid. Patients (n = 49) were women with a clinically recognized pregnancy who experienced a fetal death before 100 days' gestation. Controls (n = 409) were women who maintained a pregnancy that ended in a live birth. Homocysteine, folate, and vitamins B(6) and B(12) concentrations were measured in plasma obtained before conception. RESULTS: Mean vitamin B(6) concentration was lower in patients than in controls (34.0 versus 37.9 nmol/L, P =.04). In addition, the risk of spontaneous abortion tended to increase with decreasing plasma vitamin B(6) and folate concentration (P for trend =.06 and.07, respectively), although the significance of these trends was further reduced in logistic models that included age, body mass index, and both vitamins. The risk of spontaneous abortion was four-fold higher among women with suboptimal plasma concentrations of both folate and vitamin B(6) (folate less than or equal to 8.4 nmol/L and vitamin B(6) less than or equal to 49 nmol/L) than in those with higher plasma concentrations of both vitamins (odds ratio 4.1, 95% confidence interval 1.2, 14.4). Homocysteine and vitamin B(12) status were not associated with spontaneous abortion risk. CONCLUSION: Suboptimal preconception folate and vitamin B(6) status, especially when they occur together, may increase the risk of clinical spontaneous abortion. Additional prospective studies are needed to confirm these findings and to determine whether antenatal B vitamin supplementation reduces spontaneous abortion risk.
Plasma folate levels and risk of spontaneous abortion. George L, Mills JL, Johansson AL, Nordmark A, Olander B, Granath F, Cnattingius S. Department of Medical Epidemiology, Karolinska Institutet, PO Box 281, SE-171 77 Stockholm, Sweden.	JAMA 2002 Oct 16;288(15):1867-73 Abstract quote CONTEXT: Both folate deficiency and folic acid supplements have been reported to increase the risk of spontaneous abortion. The results are inconclusive, however, and measurements of folate have not been available in all studies. OBJECTIVE: To study the association between plasma folate levels and the risk of spontaneous abortion. DESIGN, SETTING, AND POPULATION: Population-based, matched, case-control study of case women with spontaneous abortion and control women from January 1996 through December 1998 in Uppsala County, Sweden. Plasma folate measurements were available for 468 cases and 921 controls at 6 to 12 gestational weeks. MAIN OUTCOME MEASURE: Risk of spontaneous abortion vs maternal plasma folate level. RESULTS: Compared with women with plasma folate levels between 2.20 and 3.95 ng/mL (5.0 and 8.9 nmol/L), women with low (</=2.19 ng/mL [</=4.9 nmol/L]) folate levels were at increased risk of spontaneous abortion (adjusted odds ratio [OR], 1.47; 95% confidence interval [CI], 1.01-2.14), whereas women with higher folate levels (3.96-6.16 ng/mL [9.0-13.9 nmol/L] and >/=6.17 ng/mL [>/=14.0 nmol/L]) showed no increased risk of spontaneous abortion (OR, 0.84; 95% CI, 0.59-1.20; and OR, 0.74; 95% CI, 0.47-1.16, respectively). Low folate levels were associated with a significantly increased risk when the fetal karyotype was abnormal (OR, 1.95; 95% CI, 1.09-3.48) but not when the fetal karyotype was normal (OR, 1.11; 95% CI, 0.55-2.24) or unknown (OR, 1.45; 95% CI, 0.90-2.33). CONCLUSION: Low plasma folate levels were associated with an increased risk of early spontaneous abortion.

Reference Methods
Clinical Utility
Interfering Diseases or Substances that Alter Levels
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INTERFERING DISEASES OR SUBSTANCES	CHARACTERIZATION
ALTERED LEVELS
Drugs and folate metabolism. Lambie DG, Johnson RH.	Drugs 1985 Aug;30(2):145-55 Abstract quote Folates are a group of compounds which are required in the diet and are important in DNA, amino acids and possibly also amine metabolism. The biologically active folates are in the tetrahydro form. Tetrahydrofolates are produced from unreduced dietary folates by the enzyme dihydrofolate reductase. A number of drugs such as aminopterin, methotrexate (amethopterin), pyrimethamine, trimethoprim and triamterene act as folate antagonists and produce folate deficiency by inhibiting this enzyme. With other drugs which produce low serum and tissue concentrations of folate such as anticonvulsants, antituberculosis drugs, alcohol and oral contraceptives, the mechanism of this effect is uncertain. Possible mechanism include reduced absorption, prevention of release of folate from tissue stores, altered plasma protein binding, or increased folate metabolism in the liver. Treatment with folic acid antagonists such as methotrexate readily causes megaloblastic anaemia; this can be prevented by therapy with folinic acid (5-formyltetrahydrofolate). The role of other drugs in producing megaloblastic anaemia is less certain, e.g. it occurs in less than 0.75% of patients receiving anticonvulsants. The possible neurological and psychiatric effects of folate deficiency are also uncertain. However, in patients with folate deficiency who have neuropsychiatric symptoms, neuropathy or myelopathy, and normal vitamin B12 levels, it may be of value to try therapy with folic or folinic acid.
INCREASE LEVELS
DIALYSIS
Homocysteine, vitamin B12, and serum and erythrocyte folate in peritoneal dialysis and hemodialysis patients. De Vecchi AF, Bamonti-Catena F, Finazzi S, Campolo J, Taioli E, Novembrino C, Colucci P, Accinni R, De Franceschi M, Fasano MA, Maiolo AT. Divisione di Nefrologia e Dialisi, IRCCS Ospedale Maggiore, Milano, Italy. suggests that levels of folate within the reference interval are inadequate for dialysis patients.	Perit Dial Int 2000 Mar-Apr;20(2):169-73 Abstract quote BACKGROUND: Plasma homocysteine (Hcy) is an independent risk factor for cardiovascular disease. High levels of plasma Hcy have been observed in end-stage renal disease patients. Few studies have compared peritoneal dialysis (PD) and hemodialysis (HD) patients and few data are available on erythrocyte folate (ery-F) levels in dialysis patients. OBJECTIVES: To evaluate plasma Hcy concentrations, vitamin B12 (B12), and folate status in dialysis patients; to analyze the possible causes of high Hcy levels; to follow up changes in folate and B12 concentrations after 6 months. DESIGN: A cross-sectional observational study. SETTING: Nephrology division and laboratory of hematology in a university and clinical research hospital. PATIENTS: The study included 82 patients treated with PD for 37 + 37 months and 70 patients treated with HD for 136 + 95 months. LABORATORY METHODS: Plasma Hcy was measured by the immunoenzymatic IMx Hcy FPIA method (Abbott Laboratories, Diagnostic Division, Abbott Park, IL, U.S.A.), serum folate (s-F) and ery-F by the Stratus folate fluorometric enzyme-linked assay, and B12 by the Stratus vitamin B12 fluorometric enzyme-linked assay (DADE-Behring, Newark, DE, U.S.A.). RESULTS: Ninety-six percent of PD and 97% of HD patients had Hcy levels above the cutoff (13.5 micromol/L). Homocysteine level was higher in HD than in PD patients, while the prevalence of hyperhomocysteinemia was similar with the two techniques. Erythrocyte folate was significantly higher in PD (1333 +/- 519 pmol/L) than in HD (1049 +/-511 pmol/L, p < 0.01). Statistically significant correlations were observed between Hcy and B12, s-F, ery-F, and dialysis duration. Multivariate analysis showed a strong correlation between s-F and Hcy. After 6 months there were no differences in Hcy, B12, s-F, and ery-F levels. CONCLUSIONS: Plasma Hcy levels were high in more than 95% of our dialysis patients, with no relation to the type of dialysis. Vitamin B12 and folate were normal in the majority of our patients. However, serum folate was the major determinant of Hcy levels. Such a relation between Hcy and folate suggests that levels of folate within the reference interval are inadequate for dialysis patients.
DECREASE LEVELS
HOMOCYSTEINE
Elevated serum homocysteine as a predictor for vitamin B12 or folate deficiency. Curtis D, Sparrow R, Brennan L, Van der Weyden MB. Haematology Unit, Alfred Group of Hospitals Pathology Service, Alfred Hospital, Prahran, Victoria, Australia.	Eur J Haematol 1994 Apr;52(4):227-32 Abstract quote Tissue deficiency of vitamin B12 and folate results in an increase in serum homocysteine (sHcy). We have measured sHcy in patients with reduced serum vitamin B12 and/or red cell folate (RCF) to determine its usefulness as a discriminant for the diagnostic interpretation of reduced vitamin levels. Of 3846 patients who had serum vitamin B12 and RCF assayed, 335 (9%) had reduced vitamin levels. Multivariate analysis showed a significant association between sHcy and serum creatinine (p = 0.0001), positive intrinsic factor (IF) antibody or neutrophil hypersegmentation (NHS) (p = 0.001), increased MCV (p = 0.014) and low RCF (p = 0.025) but no relationship with the level of serum vitamin B12 or haemoglobin. After censoring the patients with renal impairment (n = 54), the distribution of the remaining 72 patients with elevated sHcy was 37/151 (25%) with low serum vitamin B12 with or without low RCF and 35/130 (27%) with low RCF alone. sHcy correctly identified response to vitamin therapy in 33/35 (94%) patients who had adequate parameters to assess response. The positive predictive values of IF antibody/NHS, macrocytosis and/or low RCF for elevated sHcy were 100% and 34% respectively. Twenty-four percent of patients with a low serum vitamin B12 and elevated sHcy had no abnormal haematologic parameters as determined by the routine laboratory staff. These data suggest that the usefulness of measuring sHcy in a routine diagnostic setting is limited and a careful review of the peripheral blood for macrocytosis and NHS plus determination of RCF may be a more cost-effective process than sHcy assay in most instances to determine the presence of tissue deficiency.